CD4 T cells and monocyte/macrophages (M/M) play an important role in all phases of human immunodeficiency virus type 1 (HIV-1) infection, acting as vehicles for virus dissemination in the body and representing the major reservoir for long term persistence of HIV-1 during highly active anti-retroviral therapy (HAART) (5-7). Elimination of these viral reservoirs are an important treatment goal not achieved by current optimized highly active antiviral therapies. M/M continue to shed infectious virus, even with effective HAART therapy, and thus are a source of reinfection and evolution of treatment resistant viral strains.
Microglia, local differentiated M/M, are the main source of virus in the brain, whose pathogenic secretory products cause neuro-AIDS (8, 9). HIV-1 entry into cells occurs after binding of the viral envelope glycoprotein gp120 to specific chemokine receptors in conjunction with the CD4 receptor (10, 11) CCR5 in particular is the principal coreceptor for the HIV-1 strains that are transmitted between individuals and which predominate during the early years of infection and predominate in the brain where they cause the manifestation of neuro-AIDS, via infection of CCR5 expressing monocytes and microglia (12, 13). M/M and microglia are infected primarily by CCR5 using HIV strains. CCR5 is also expressed on neuronal cell lines and astrocytes in the brain and, whereas it is known that neuronal cells are usually not productively infected by HIV-1, in vitro studies have shown that natural ligands of CCR5 protect neurons from gp120-mediated apoptosis (14-16). Drugs which block CCR5 receptors on brain cells would be useful treatments to prevent neuronal apoptosis, a cause of Neuro-AIDS and dementia or mental impairment. Furthermore antibodies that block gp120 binding to CCR5 would be useful as a broadly neutralizing AIDS vaccine as CCR5 co-receptor use is an invariant feature of viral strains that establish initial infection.
Dala1-peptide T-amide (DAPTA) is a synthetic peptide derived from HIV gp120 that functions as a viral entry inhibitor by blocking gp120 binding to CCR5 (13, 17, 18). Freshly prepared, compared to stored, solutions of this small peptide suppresses the infection of peripheral blood monocytes in vitro (17) suggesting that blocking infection of monocytes in patients would prevent and reduce the population of infected differentiated M/M which are resistant to current treatments and which form a reservoir of infected differentiated M/M in patients MM reservoirs are sources of infection for T cells which sustain low level viral replication in the face of therapy leading to resistance development and treatment failure. Current HAART therapies do not effectively treat the monocyte.macrophage reservoirs (2, 19, 20).
A small clinical trial of DAPTA has shown mixed results, having some antiviral benefits in the monocytes, but failing to reduce plasma viral load, the main endpoints of the study. An earlier multi-site, placebo-controlled trial conducted by the NIH had also failed to achieve significance on the main trial endpoints, neurocognitive benefits (21), as did a placebo-controlled trial of peptide T for HIV associated neuropathhic pain (22). An analysis of frozen stored plasma samples conducted by the NIMH in the early-1990's from the randomized double-blind placebo-controlled trial of DAPTA for HIV-associated cognitive impairment (21) found a barely significant reduction in viral load (0.54 log 10, p=. 037), a modest effect, not comparable to the −2 log 10 reductions of viral load offered by several current anti-viral therapies. These marginal clinical benefits were inexplicable in the context of the many successful in vitro studies and an explanation was sought. Patient reports of sporadic gel foiniation in trial nasal sprayers suggested an explanation focused on the bio-physical character of the peptide drug. This sporadic change in physical state was unexpected and had not been revealed in many scientific studies which had used peptide T, and had not been reported in any of the clinical articles. An analysis of clinical trial formulated drug according to U.S. Pat. Nos. 5,276,016 and 5,834,429 revealed substantial aggregation and loss of biological activity, in a variable manner, dependent upon individual storage conditions and temperature. Further direct experimentation showed that DAPTA and peptide T quickly formed aggreagates in solution, which over time formed fibrils, and that both aggregates and fibril forms were devoid of biological activity. Therefore reduction to practice required that the non-obvious formation of multimers must be remedied for considerations of both patient safety and drug efficacy.